Chemically modified mRNA is capable of inducing therapeutic levels of protein expression while circumventing the threat of genomic integration often associated with viral vectors. We utilized this novel therapeutic tool to express the regulatory T cell transcription factor, FOXP3, in a time- and site-specific fashion in murine lung, in order to prevent allergic asthma in vivo. We show that modified Foxp3 mRNA rebalanced pulmonary T helper cell responses and protected from allergen-induced tissue inflammation, airway hyperresponsiveness, and goblet cell metaplasia in 2 asthma models. This protection was conferred following delivery of modified mRNA either before or after the onset of allergen challenge, demonstrating its potential as both a preventive and a therapeutic agent. Mechanistically, FOXP3 induction controlled Th2 and Th17 inflammation by regulating innate immune cell recruitment through an IL-10–dependent pathway. The protective effects of FOXP3 could be reversed by depletion of IL-10 or administration of recombinant IL-17A or IL-23. Delivery of Foxp3 mRNA to sites of inflammation may offer a novel, safe therapeutic tool for the treatment of allergic asthma and other diseases driven by an imbalance in helper T cell responses.
Hypomorphic mutations in the bone morphogenic protein receptor (BMPR2) confer a much greater risk for developing pulmonary arterial hypertension (PAH). However, not all carriers of a mutation in the BMPR2 gene suffer from PAH. We have previously shown that prolonged T helper 2 (Th2) responses in the lungs to a mild antigen delivered via the airways induce severe pulmonary arterial remodeling, but no pulmonary hypertension. The current studies were designed to test the idea that Th2 responses to a mild antigen together with the expression of a hypomorphic BMPR2 gene would trigger pulmonary hypertension.
Mice that expressed a hypomorphic BMPR2 transgene (transgene-positive) and transgene-negative mice were either exposed to saline, or primed and exposed to a mild antigen (Ovalbumin) over a prolonged period of time. Only transgene-positive but not transgene-negative mice exposed to antigen developed significantly increased right ventricular systolic pressures, while both groups showed pulmonary artery remodeling with severe muscularization and airway inflammation to a similar degree. Antigen exposure resulted in a smaller increase in the percentage of Interleukin (IL)-13 positive T cells in the lymph nodes, and in a smaller increase in resistin-like-molecule (RELM)α expression and a decreased ratio of expression of IL-33 relative to its receptor (IL-1-receptor-like 1, IL1RL1-ST2) in the right ventricles of transgene-positive mice compared to transgene-negative animals. Furthermore, only antigen-challenged transgene-positive mice showed a significant increase in Interferon (IFN)γ positive T cells over saline-exposed controls.
Our study suggests that exposure with a mild Th2 antigen can trigger pulmonary hypertension on the background of the expression of a hypomorphic BMPR2 gene and that conversely, the expression of the hypomorphic BMPR2 gene can alter the immune response to a mild, inhaled antigen.
IL-18 plays a key role in the pathogenesis of pulmonary inflammatory diseases including pulmonary infection, pulmonary fibrosis, lung injury and chronic obstructive pulmonary disease (COPD). However, it is unknown whether IL-18 plays any role in the pathogenesis of asthma. We hypothesized that overexpression of mature IL-18 protein in the lungs may exacerbate disease activities of asthma. We established lung-specific IL-18 transgenic mice on a Balb/c genetic background. Female mice sensitized– and challenged– with antigen (ovalbumin) were used as a mouse asthma model. Pulmonary inflammation and emphysema were not observed in the lungs of naïve transgenic mice. However, airway hyperresponsiveness and airway inflammatory cells accompanied with CD4+ T cells, CD8+ T cells, eosinophils, neutrophils, and macrophages were significantly increased in ovalbumin-sensitized and challenged transgenic mice, as compared to wild type Balb/c mice. We also demonstrate that IL-18 induces IFN-γ, IL-13, and eotaxin in the lungs of ovalbumin-sensitized and challenged transgenic mice along with an increase in IL-13 producing CD4+ T cells. Treatment with anti-CD4 monoclonal antibody or deletion of the IL-13 gene improves ovalbumin-induced airway hyperresponsiveness and reduces airway inflammatory cells in transgenic mice. Overexpressing the IL-18 protein in the lungs induces type 1 and type 2 cytokines and airway inflammation, and results in increasing airway hyperresponsiveness via CD4+ T cells and IL-13 in asthma.
Despite its incorporation into research studies, the safety aspects of segmental allergen bronchoprovocation and differences in cellular response among different allergens have received limited consideration.
We performed 87 segmental challenges in 77 allergic asthma subjects. Allergen dose was based on each subject’s response to whole lung allergen challenge. Bronchoalveolar lavage was performed at 0 and 48 hours. Safety indicators included spirometry, oxygen saturation, heart rate, and symptoms.
Among subjects challenged with ragweed, cat dander, or house dust mite, there were no differences in safety indicators. Subjects demonstrated a modest oxygen desaturation and tachycardia during the procedure that returned to normal prior to discharge. We observed a modest reduction in forced vital capacity and forced expiratory volume in one second following bronchoscopy. The most common symptoms following the procedure were cough, sore throat and fatigue. Total bronchoalveolar lavage fluid cell numbers increased from 13±4 to 106±108×104 per milliliter and eosinophils increased from 1±2 to 44±20 percent, with no significant differences among the three allergens.
In mild allergic asthma, segmental allergen bronchoprovocation, using individualized doses of aeroallergens, was safe and yielded similar cellular responses.
The pollen coat is the first structure of the pollen to encounter the mucosal immune system upon inhalation. Prior characterizations of pollen allergens have focused on water-soluble, cytoplasmic proteins, but have overlooked much of the extracellular pollen coat. Due to washing with organic solvents when prepared, these pollen coat proteins are typically absent from commercial standardized allergenic extracts (i.e., “de-fatted”), and, as a result, their involvement in allergy has not been explored.
Using a unique approach to search for pollen allergenic proteins residing in the pollen coat, we employed transmission electron microscopy (TEM) to assess the impact of organic solvents on the structural integrity of the pollen coat. TEM results indicated that de-fatting of Cynodon dactylon (Bermuda grass) pollen (BGP) by use of organic solvents altered the structural integrity of the pollen coat. The novel IgE-binding proteins of the BGP coat include a cysteine protease (CP) and endoxylanase (EXY). The full-length cDNA that encodes the novel IgE-reactive CP was cloned from floral RNA. The EXY and CP were purified to homogeneity and tested for IgE reactivity. The CP from the BGP coat increased the permeability of human airway epithelial cells, caused a clear concentration-dependent detachment of cells, and damaged their barrier integrity.
Using an immunoproteomics approach, novel allergenic proteins of the BGP coat were identified. These proteins represent a class of novel dual-function proteins residing on the coat of the pollen grain that have IgE-binding capacity and proteolytic activity, which disrupts the integrity of the airway epithelial barrier. The identification of pollen coat allergens might explain the IgE-negative response to available skin-prick-testing proteins in patients who have positive symptoms. Further study of the role of these pollen coat proteins in allergic responses is warranted and could potentially lead to the development of improved diagnostic and therapeutic tools.
Imaging studies have demonstrated that ventilation during bronchoconstriction in subjects with asthma is patchy with large ventilation defective areas (Vdefs). Based on a theoretical model, we postulated that during bronchoconstriction, as smooth muscle force activation increases, a patchy distribution of ventilation should emerge, even in the presence of minimal heterogeneity the lung. We therefore theorized that in normal lungs, Vdefs should also emerge in regions of the lung with reduced expansion.
We studied 12 healthy subjects to evaluate whether Vdefs formed during bronchoconstriction, and compared their Vdefs with those observed in 9 subjects with mild asthma.
Spirometry, low frequency (0.15 Hz) lung elastance and resistance, and regional ventilation by intravenous 13NN-saline positron emission tomography were measured before and after a challenge with nebulized methacholine. Vdefs were defined as regions with elevated residual 13NN after a period of washout. The average location, ventilation, volume, and fractional gas content of the Vdefs, relative to those of the rest of the lung, were calculated for both groups.
Consistent with the predictions of the theoretical model, both healthy subjects and those with asthma developed Vdefs. These Vdefs tended to form in regions that, at baseline, had a lower degree of lung inflation and, in healthy subjects, tended to occur in more dependent locations than in subjects with asthma.
The formation of Vdefs is determined by the state of inflation prior to bronchoconstriction.
Sepsis remains a major cause of mortality in intensive care units, better therapies are urgently needed. Gram-negative bacterial lipopolysaccharide (LPS) is an important trigger of sepsis. We have demonstrated that berberine (Ber) protects against lethality induced by LPS, which is enhanced by yohimbine (Y) pretreatment, and Ber combined with Y also improves survival in septic mice. However, the precise mechanisms by which Y enhances protection of Ber against LPS - induced lethality remain unclear. The present study confirmed that simultaneously administered Y also enhanced protection of Ber against LPS-induced lethality. Ber or/and Y attenuated liver injury, but not renal injury in LPS-challenged mice. Ber or/and Y all inhibited LPS-stimulated IκBα, JNK and ERK phosphorylation, NF-κB activation as well as TNF-α production. Ber also increased IL-10 production in LPS-challenged mice, which was enhanced by Y. Furthermore, Ber or/and Y all suppressed LPS-induced IRF3, TyK2 and STAT1 phosphorylation, as well as IFN-β and IP-10 mRNA expression in spleen of mice at 1 h after LPS challenge. Especially, Y enhanced the inhibitory effect of Ber on LPS-induced IP-10 mRNA expression. In vitro experiments further demonstrated that Y significantly enhanced the inhibitory effect of Ber on TNF-α production in LPS-treated peritoneal macrophages, Ber combined with Y promoted LPS-induced IL-10 production and LPS-stimulated IκBα, JNK, ERK and IRF3 phosphorylation and NF-κB activation were also suppressed by Ber or/and Y pretreatment in peritoneal macrophages. Taken together, these results demonstrate that Y enhances the protection of Ber against LPS-induced lethality in mice via attenuating liver injury, upregulating IL-10 production and suppressing IκBα, JNK, ERK and IRF3 phosphorylation. Ber combined with Y may be an effective immunomodulator agent for the prevention of sepsis.
Peanut allergy is the leading cause of deaths due to food-induced anaphylaxis but despite continued research, there are currently no specific treatments available. Challenge testing is limited in patients due to the high risk of adverse reactions, emphasising the need for an appropriate animal model. In the present study we examine the induction of allergic responses in a sheep model for peanut allergy. Sheep were sensitised with peanut (PN) extract and in separate injections with ovalbumin (OVA) or house dust mite (HDM) extract. Serum PN-specific IgE responses were detected in 40–50% of immunised sheep, while only 10% (1 of 10 sheep) showed detectable OVA-specific IgE. All PN-allergic sheep tested showed an Ara h 1-specific IgE response, while four out of five allergic sheep showed an Ara h 2-specific IgE response. Animals with high serum IgE levels to HDM were also PN IgE-positive. Of the PN-sensitised animals with high PN-specific IgE, 80% also showed an immediate hypersensitivity reaction following an intradermal PN injection. This new large animal model of peanut allergy may provide a useful tool for future investigations of allergen-associated immune mechanisms and specific immunotherapy.
Accumulating evidence suggests a contribution of T cell-derived IL-17, IL-21 and IL-22 cytokines in skin immune homeostasis as well as inflammatory disorders. Here, we analyzed whether the cytokine-producing T lymphocytes could be induced by the different subsets of human skin dendritic cells (DCs), i.e., epidermal Langerhans cells (LCs), dermal CD1c+CD14− and CD14+ DCs (DDCs). DCs were purified following a 2-day migration from separated epidermal and dermal sheets and co-cultured with allogeneic T cells before cytokine secretion was explored. Results showed that no skin DCs could induce substantial IL-17 production by naïve CD4+ or CD8+T lymphocytes whereas all of them could induce IL-17 production by memory T cells. In contrast, LCs and CD1c+CD14−DDCs were able to differentiate naïve CD4+T lymphocytes into IL-22 and IL-21-secreting cells, LCs being the most efficient in this process. Intracellular cytokine staining showed that the majority of IL-21 or IL-22 secreting CD4+T lymphocytes did not co-synthesized IFN-γ, IL-4 or IL-17. IL-21 and IL-22 production were dependent on the B7/CD28 co-stimulatory pathway and ICOS-L expression on skin LCs significantly reduced IL-21 level. Finally, we found that TGF-β strongly down-regulates both IL-21 and IL-22 secretion by allogeneic CD4+ T cells. These results add new knowledge on the functional specialization of human skin DCs and might suggest new targets in the treatment of inflammatory skin disorders.
The contraction phase of antigen-specific immune responses involves the apoptotic loss of numerous activated lymphocytes. While apoptotic cells are known to induce immune suppression, the mechanisms involved therein are still ambiguous. Some reports have speculated that macrophages can induce regulatory T cells (Tregs) after engulfing apoptotic cells. In this study, we showed that dendritic cells (DCs) that phagocytose apoptotic T cells acquire inhibitory function (named DCapos) toward CD4+ and CD8+ T cells. These inhibitory DCs could not induce the generation of Tregs, but they were found to directly inhibit mDCs that initiate CD4+ and CD8+ T cell proliferation both in vitro and in vivo. Soluble factors including NO play a role in the DCapos-induced suppression of CD4+ and CD8+ T cell proliferation. Further results showed that STAT3 phosphorylation and inducible nitric oxide synthase (iNOS) generation were enhanced when DCs were co-cultured with apoptotic cells. Both iNOS transcription and NO secretion were inhibited in the presence of the specific p-STAT3 inhibitor JSI-124. All the data indicated that apoptotic cells could turn DCs to inhibitory DCs, which might play important roles in the suppression of immune responses. STAT3 activation and the consequent release of NO are responsible for the inhibitory functions of DCapos.
The cannabinoid system is known to be involved in the regulation of inflammatory processes. Therefore, drugs targeting cannabinoid receptors are considered as candidates for anti-inflammatory and tissue protective therapy. We demonstrated that the prototypical cannabinoid agonist R(+)WIN55,212-2 (WIN) reduced the secretion of matrix metalloproteinase-9 (MMP-9) in a murine model of cigarette-smoke induced lung inflammation. In experiments using primary cells and cell lines of the monocyte-macrophage-system we found that binding of the cannabinoid-receptor agonist WIN to a stereo-selective, specific binding site in cells of the monocyte-macrophage-system induced a significant down-regulation of MMP-9 secretion and disturbance of intracellular processing, which subsequently down-regulated MMP-9 mRNA expression via a ERK1/2-phosphorylation-dependent pathway. Surprisingly, the anti-inflammatory effect was independent from classical cannabinoid receptors. Our experiments supposed an involvement of TRPV1, but other yet unidentified sites are also possible. We conclude that cannabinoid-induced control of MMP-9 in the monocyte-macrophage system via a cannabinoid-receptor independent pathway represents a general option for tissue protection during inflammation, such as during lung inflammation and other diseases associated with inflammatory tissue damage.
Clinical interpretation of bronchoalveolar lavage fluid results is dependent on the availability of reference values for healthy individuals. Only a few studies have published such reference values and the applicability of results is restricted by small sample sizes and the limited representativeness of the study population. We aim to investigate the influence of age, gender, collection site and season on bronchoalveolar lavage fluid results and to establish reference values for use in clinical practice.
Bronchoalveolar lavage fluid data from 295 healthy never-smoking volunteers, investigated during 1990–2009, were analyzed retrospectively. 47 volunteers had 2–5 repeat lavages during the course of several years. Fluid recovery, total number of cells, cell concentration, and differential cell counts on cytospin prepared slides were recorded. Reference values, as represented by the 5th to the 95th percentile, were 72–96% for macrophages, 2–26% for lymphocytes, 0–4% for neutrophils and 0–1% for eosinophils. Basophils and mast cells were rare. When repeat lavages were performed, there was a relatively large intra-individual variability, mainly for macrophages and lymphocytes. An age dependent decrease of lavage fluid return was present, but there was no age dependent correlation with any of the other BALF parameters. The BALF cell parameters were independent of gender, season and site (lingula vs. middle lobe).
Our data show that bronchoalveolar lavage fluid cell differential count is independent of age, gender, season and collection site (RML or lingua). It therefore seems acceptable to use the same reference values for all never-smoking individuals.
Increasing incidence and substantial morbidity and mortality of respiratory diseases requires the development of new human-specific anti-inflammatory and disease-modifying therapeutics. Therefore, new predictive animal models that closely reflect human lung pathology are needed. In the current study, a tiered acute lipopolysaccharide (LPS)-induced inflammation model was established in marmoset monkeys (Callithrix jacchus) to reflect crucial features of inflammatory lung diseases. Firstly, in an ex vivo approach marmoset and, for the purposes of comparison, human precision-cut lung slices (PCLS) were stimulated with LPS in the presence or absence of the phosphodiesterase-4 (PDE4) inhibitor roflumilast. Pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and macrophage inflammatory protein-1 beta (MIP-1β) were measured. The corticosteroid dexamethasone was used as treatment control. Secondly, in an in vivo approach marmosets were pre-treated with roflumilast or dexamethasone and unilaterally challenged with LPS. Ipsilateral bronchoalveolar lavage (BAL) was conducted 18 hours after LPS challenge. BAL fluid was processed and analyzed for neutrophils, TNF-α, and MIP-1β. TNF-α release in marmoset PCLS correlated significantly with human PCLS. Roflumilast treatment significantly reduced TNF-α secretion ex vivo in both species, with comparable half maximal inhibitory concentration (IC50). LPS instillation into marmoset lungs caused a profound inflammation as shown by neutrophilic influx and increased TNF-α and MIP-1β levels in BAL fluid. This inflammatory response was significantly suppressed by roflumilast and dexamethasone. The close similarity of marmoset and human lungs regarding LPS-induced inflammation and the significant anti-inflammatory effect of approved pharmaceuticals assess the suitability of marmoset monkeys to serve as a promising model for studying anti-inflammatory drugs.
Cigarette smoke exposure is strongly associated with airway hyperreactivity (AHR) which is the main characteristic seen in asthma. The intracellular MAPK signaling pathways are suggested to be associated with the airway damage to the AHR. In the present study, we hypothesize that secondhand cigarette smoke (SHS) exposure upregulates the bronchial contractile receptors via activation of the Raf/ERK/MAPK pathway.
Rats were exposed to SHS for 3 h daily for up to 8 weeks. The receptor agonists-induced bronchial contractile reactivity was analyzed with a sensitive myograph system. The mRNA transcription and protein translation of the target receptors and the kinases in Raf/ERK/MAPK pathway were investigated by real-time PCR, Western blotting and immunofluorescence, respectively. Compared with exposure to fresh air, SHS induced enhanced bronchial contractile responses mediated by the 5-hydroxytryptamine 2A (5-HT2A) receptors as well as the endothelin type B (ETB) and type A (ETA) receptors. The response curves were shifted toward the left with an increased maximal contraction (Emax) demonstrating that SHS induced AHR. Additionally, the mRNA and protein levels of the 5-HT2A, ETB and ETA receptors were increased. Furthermore, SHS exposure increased the phosphorylation of Raf-1 and ERK1/2, but it did not alter p38 or JNK. A Raf-1 inhibitor (GW5074) suppressed the SHS-induced increase in the expression of 5-HT2A and ETA receptors and the receptor-mediated AHR.
Our findings show that SHS exposure induces transcriptional upregulation of the 5-HT2A, ETB and ETA receptors in rat bronchial smooth muscle cells, which mediates AHR. The Raf/ERK/MAPK pathway is involved in SHS-associated receptor upregulation and AHR.
Viral and bacterial respiratory tract infections in early-life are linked to the development of allergic airway inflammation and asthma. However, the mechanisms involved are not well understood. We have previously shown that neonatal and infant, but not adult, chlamydial lung infections in mice permanently alter inflammatory phenotype and physiology to increase the severity of allergic airway disease by increasing lung interleukin (IL)-13 expression, mucus hyper-secretion and airway hyper-responsiveness. This occurred through different mechanisms with infection at different ages. Neonatal infection suppressed inflammatory responses but enhanced systemic dendritic cell:T-cell IL-13 release and induced permanent alterations in lung structure (i.e., increased the size of alveoli). Infant infection enhanced inflammatory responses but had no effect on lung structure. Here we investigated the role of hematopoietic cells in these processes using bone marrow chimera studies.
Neonatal (<24-hours-old), infant (3-weeks-old) and adult (6-weeks-old) mice were infected with C. muridarum. Nine weeks after infection bone marrow was collected and transferred into recipient age-matched irradiated naïve mice. Allergic airway disease was induced (8 weeks after adoptive transfer) by sensitization and challenge with ovalbumin. Reconstitution of irradiated naïve mice with bone marrow from mice infected as neonates resulted in the suppression of the hallmark features of allergic airway disease including mucus hyper-secretion and airway hyper-responsiveness, which was associated with decreased IL-13 levels in the lung. In stark contrast, reconstitution with bone marrow from mice infected as infants increased the severity of allergic airway disease by increasing T helper type-2 cell cytokine release (IL-5 and IL-13), mucus hyper-secretion, airway hyper-responsiveness and IL-13 levels in the lung. Reconstitution with bone marrow from infected adult mice had no effects.
These results suggest that an infant chlamydial lung infection results in long lasting alterations in hematopoietic cells that increases the severity of allergic airway disease in later-life.
Epidemiological studies suggest an association between exposure to volatile organic compounds (VOCs) and adverse allergic and respiratory symptoms. However, whether VOCs exhibit a causal role as adjuvants in asthma development remains unclear.
To investigate the effect of VOC exposure on the development of allergic airway inflammation Balb/c mice were exposed to VOCs emitted by new polyvinylchloride (PVC) flooring, sensitized with ovalbumin (OVA) and characterized in acute and chronic murine asthma models. Furthermore, prevalent evaporated VOCs were analyzed and mice were exposed to selected single VOCs.
Exposure of mice to PVC flooring increased eosinophilic lung inflammation and OVA-specific IgE serum levels compared to un-exposed control mice. The increased inflammation was associated with elevated levels of Th2-cytokines. Long-term exposure to PVC flooring exacerbated chronic airway inflammation. VOCs with the highest concentrations emitted by new PVC flooring were N-methyl-2-pyrrolidone (NMP) and 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB). Exposure to NMP or TXIB also increased the allergic immune response in OVA-sensitized mice. In vitro or in vivo exposure to NMP or TXIB reduced IL-12 production in maturing dendritic cells (DCs) and enhanced airway inflammation after adoptive DC transfer into Balb/c mice. At higher concentrations both VOCs induced oxidative stress demonstrated by increased isoprostane and glutathione-S-transferase-pi1 protein levels in the lung of non-sensitized mice. Treatment of PVC flooring-exposed mice with N-acetylcysteine prevented the VOC-induced increase of airway inflammation.
Our results demonstrate that exposure to VOCs may increase the allergic immune response by interfering with DC function and by inducing oxidative stress and has therefore to be considerate as risk factor for the development of allergic diseases.
Previous work from our laboratory demonstrated that IL-4Rα expression on a myeloid cell type was responsible for enhancement of Th2-driven eosinophilic inflammation in a mouse model of allergic lung inflammation. Subsequently, we have shown that IL-4 signaling through type I IL-4 receptors on monocytes/macrophages strongly induced activation of the IRS-2 pathway and a subset of genes characteristic of alternatively activated macrophages. The direct effect(s) of IL-4 and IL-13 on mouse eosinophils are not clear. The goal of this study was determine the effect of IL-4 and IL-13 on mouse eosinophil function.
Standard Transwell chemotaxis assay was used to assay migration of mouse eosinophils and signal transduction was assessed by Western blotting.
Here we determined that (i) mouse eosinophils express both type I and type II IL-4 receptors, (ii) in contrast to human eosinophils, mouse eosinophils do not chemotax to IL-4 or IL-13 although (iii) pre-treatment with IL-4 but not IL-13 enhanced migration to eotaxin-1. This IL-4-mediated enhancement was dependent on type I IL-4 receptor expression: γC-deficient eosinophils did not show enhancement of migratory capacity when pre-treated with IL-4. In addition, mouse eosinophils responded to IL-4 with the robust tyrosine phosphorylation of STAT6 and IRS-2, while IL-13-induced responses were considerably weaker.
The presence of IL-4 in combination with eotaxin-1 in the allergic inflammatory milieu could potentiate infiltration of eosinophils into the lungs. Therapies that block IL-4 and chemokine receptors on eosinophils might be more effective clinically in reducing eosinophilic lung inflammation.
Reduced physical activity is an important feature of Chronic Obstructive Pulmonary Disease (COPD). Various activity monitors are available but their validity is poorly established. The aim was to evaluate the validity of six monitors in patients with COPD. We hypothesized triaxial monitors to be more valid compared to uniaxial monitors. Thirty-nine patients (age 68±7years, FEV1 54±18%predicted) performed a one-hour standardized activity protocol. Patients wore 6 monitors (Kenz Lifecorder (Kenz), Actiwatch, RT3, Actigraph GT3X (Actigraph), Dynaport MiniMod (MiniMod), and SenseWear Armband (SenseWear)) as well as a portable metabolic system (Oxycon Mobile). Validity was evaluated by correlation analysis between indirect calorimetry (VO2) and the monitor outputs: Metabolic Equivalent of Task [METs] (SenseWear, MiniMod), activity counts (Actiwatch), vector magnitude units (Actigraph, RT3) and arbitrary units (Kenz) over the whole protocol and slow versus fast walking. Minute-by-minute correlations were highest for the MiniMod (r = 0.82), Actigraph (r = 0.79), SenseWear (r = 0.73) and RT3 (r = 0.73). Over the whole protocol, the mean correlations were best for the SenseWear (r = 0.76), Kenz (r = 0.52), Actigraph (r = 0.49) and MiniMod (r = 0.45). The MiniMod (r = 0.94) and Actigraph (r = 0.88) performed better in detecting different walking speeds. The Dynaport MiniMod, Actigraph GT3X and SenseWear Armband (all triaxial monitors) are the most valid monitors during standardized physical activities. The Dynaport MiniMod and Actigraph GT3X discriminate best between different walking speeds.
Glutaredoxins (Grx) are redox enzymes that remove glutathione bound to protein thiols, know as S-glutathionylation (PSSG). PSSG is a reservoir of GSH and can affect the function of proteins. It inhibits the NF-κB pathway and LPS aspiration in Grx1 KO mice with decreased inflammatory cytokine levels. In this study we investigated whether absence of Grx1 similarly repressed cigarette smoke-induced inflammation in an exposure model in mice. Cigarette smoke exposure for four weeks decreased lung PSSG levels, but increased PSSG in lavaged cells and lavage fluid (BALF). Grx1 KO mice had increased levels of PSSG in lung tissue, BALF and BAL cells in response to smoke compared to wt mice. Importantly, levels of multiple inflammatory mediators in the BALF were decreased in Grx1 KO animals following cigarette smoke exposure compared to wt mice, as were levels of neutrophils, dendritic cells and lymphocytes. On the other hand, macrophage numbers were higher in Grx1 KO mice in response to smoke. Although cigarette smoke in vivo caused inverse effects in inflammatory and resident cells with respect to PSSG, primary macrophages and epithelial cells cultured from Grx1 KO mice both produced less KC compared to cells isolated from WT mice after smoke extract exposure. In this manuscript, we provide evidence that Grx1 has an important role in regulating cigarette smoke-induced lung inflammation which seems to diverge from its effects on total PSSG. Secondly, these data expose the differential effect of cigarette smoke on PSSG in inflammatory versus resident lung cells.
The mammalian target of rapamycin (mTOR) modulates immune responses and cellular proliferation. The objective of this study was to assess whether inhibition of mTOR with rapamycin modifies disease severity in two experimental murine models of house dust mite (HDM)-induced asthma. In an induction model, rapamycin was administered to BALB/c mice coincident with nasal HDM challenges for 3 weeks. In a treatment model, nasal HDM challenges were performed for 6 weeks and rapamycin treatment was administered during weeks 4 through 6. In the induction model, rapamycin significantly attenuated airway inflammation, airway hyperreactivity (AHR) and goblet cell hyperplasia. In contrast, treatment of established HDM-induced asthma with rapamycin exacerbated AHR and airway inflammation, whereas goblet cell hyperplasia was not modified. Phosphorylation of the S6 ribosomal protein, which is downstream of mTORC1, was increased after 3 weeks, but not 6 weeks of HDM-challenge. Rapamycin reduced S6 phosphorylation in HDM-challenged mice in both the induction and treatment models. Thus, the paradoxical effects of rapamycin on asthma severity paralleled the activation of mTOR signaling. Lastly, mediastinal lymph node re-stimulation experiments showed that treatment of rapamycin-naive T cells with ex vivo rapamycin decreased antigen-specific Th2 cytokine production, whereas prior exposure to in vivo rapamycin rendered T cells refractory to the suppressive effects of ex vivo rapamycin. We conclude that rapamycin had paradoxical effects on the pathogenesis of experimental HDM-induced asthma. Thus, consistent with the context-dependent effects of rapamycin on inflammation, the timing of mTOR inhibition may be an important determinant of efficacy and toxicity in HDM-induced asthma.
The nucleotide adenosine-5′-monophosphate (AMP) can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A1 and A2a receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC) function. AMP increased intracellular Ca2+ concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A1 receptors. AMP strongly inhibited secretion of TNF-α and IL-12p70, while it enhanced production of IL-10 both via activation of A2a receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4+CD45RA+ T cells inhibited IFN-γ production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic naïve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5′-(alpha,beta-methylene) diphosphate (APCP). Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA) was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders.
Mesenchymal stromal cells (MSCs) reside in many organs including lung, as shown by their isolation from fetal lung tissues, bronchial stromal compartment, bronchial-alveolar lavage and transplanted lung tissues. It is still controversial whether lung MSCs can undergo mesenchymal-to-epithelial-transition (MET) and possess immune regulatory properties. To this aim, we isolated, expanded and characterized MSCs from normal adult human lung (lung-hMSCs) and compared with human bone marrow-derived MSCs (BM-hMSCs). Our results show that lung-MSCs reside at the perivascular level and do not significantly differ from BM-hMSCs in terms of immunophenotype, stemness gene profile, mesodermal differentiation potential and modulation of T, B and NK cells. However, lung-hMSCs express higher basal level of the stemness-related marker nestin and show, following in vitro treatment with retinoic acid, higher epithelial cell polarization, which is anyway partial when compared to a control epithelial bronchial cell line. Although these results question the real capability of acquiring epithelial functions by MSCs and the feasibility of MSC-based therapeutic approaches to regenerate damaged lung tissues, the characterization of this lung-hMSC population may be useful to study the involvement of stromal cell compartment in lung diseases in which MET plays a role, such as in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.
Smoking is a risk factor for various lung diseases in which BAL may be used as a part of a clinical investigation. Interpretation of BAL fluid cellularity is however difficult due to high variability, in particular among smokers. In this study we aimed to evaluate the effect of smoking on BAL cellular components in asymptomatic smokers. The effects of smoking cessation, age and gender were also investigated in groups of smokers and exsmokers.
We performed a retrospective review of BAL findings, to our knowledge the largest single center investigation, in our department from 1999 to 2009. One hundred thirty two current smokers (48 males and 84 females) and 44 ex-smokers (16 males and 28 females) were included. A group of 295 (132 males and 163 females) never-smokers served as reference.
The median [5–95 pctl] total number of cells and cell concentration in current smokers were 63.4 [28.6–132.1]×106 and 382.1 [189.7–864.3]×106/L respectively and correlated positively to the cumulative smoking history. Macrophages were the predominant cell type (96.7% [90.4–99.0]) followed by lymphocytes (2% [0.8–7.7]) and neutrophils (0.6% [0–2.9]). The concentration of all inflammatory cells was increased in smokers compared to never smokers and ex-smokers. BAL fluid recovery was negatively correlated with age (p<0.001). Smoking men had a lower BAL fluid recovery than smoking women.
Smoking has a profound effect on BAL fluid cellularity, which is dependent on smoking history. Our results performed on a large group of current smokers and ex-smokers in a well standardized way, can contribute to better interpretation of BAL fluid cellularity in clinical context.
In a mouse model of viral induced atopic disease, expression of FcεRI on dendritic cells is critical. While adult human conventional (cDC) and plasmacytoid (pDC) dendritic cells have been shown to express FcεRI, it is not known if this receptor is expressed in childhood and how its expression is governed by IgE.
Following informed consent of subjects (n = 27, aged 12–188 months), peripheral blood was stained for surface expression of CD19, ILT7, CD1c, IgE, FcεRI and analyzed by flow cytometry (cDC: CD19− ILT7− CD1c+; pDC: CD19− ILT7+ CD1c−). Total and specific serum IgE levels to food and inhalant allergens were determined by ImmunoCAP, and the relationship between FcεRI expression on dendritic cells and sensitization, free IgE, cell bound IgE, and age was determined.
Independent of sensitization status, FcεRI expression was noted on cDC and pDC as early as 12 months of age. Serum IgE level correlated with expression of FcεRI on cDC, but not pDC. Based on the concentration of IgE, a complex relationship was found between surface bound IgE and expression of FcεRI on cDC. pDC exhibited a linear relationship of FcεRI expression and bound IgE that was consistent through all IgE concentrations.
In children, FcεRI expression on cDC and pDC is modulated differently by serum and cell bound IgE. IgE governance of FcεRI expression on cDC depends upon a complex relationship. Further studies are needed to determine the functional roles of FcεRI on cDC and pDC.
Food allergy may affect the gastrointestinal tract and eosinophilia is often associated with allergic gastrointestinal disorders. Allergy to peanuts is a life-threatening condition and effective and safe treatments still need to be developed. The present study aimed to evaluate the effects of sustained oral exposure to peanuts on the esophageal and jejunal mucosa in sensitized mice. We also evaluated the effects of desensitization with epicutaneous immunotherapy (EPIT) on these processes.
Mice were sensitized by gavages with whole peanut protein extract (PPE) given with cholera toxin. Sensitized mice were subsequently exposed to peanuts via a specific regimen and were then analysed for eosinophilia in the esophagus and gut. We also assessed mRNA expression in the esophagus, antibody levels, and peripheral T-cell response. The effects of EPIT were tested when intercalated with sensitization and sustained oral peanut exposure.
Sustained oral exposure to peanuts in sensitized mice led to severe esophageal eosinophilia and intestinal villus sub-atrophia, i.e. significantly increased influx of eosinophils into the esophageal mucosa (136 eosinophils/mm2) and reduced villus/crypt ratios (1.6±0.15). In the sera, specific IgE levels significantly increased as did secretion of Th2 cytokines by peanut-reactivated splenocytes. EPIT of sensitized mice significantly reduced Th2 immunological response (IgE response and splenocyte secretion of Th2 cytokines) as well as esophageal eosinophilia (50 eosinophils/mm2, p<0.05), mRNA expression of Th2 cytokines in tissue - eotaxin (p<0.05), IL-5 (p<0.05), and IL-13 (p<0.05) -, GATA-3 (p<0.05), and intestinal villus sub-atrophia (2.3±0.15). EPIT also increased specific IgG2a (p<0.05) and mRNA expression of Foxp3 (p<0.05) in the esophageal mucosa.
Gastro-intestinal lesions induced by sustained oral exposure in sensitized mice are efficaciously treated by allergen specific EPIT.